Recent Advances in the Understanding of HIV-1 Latency.

An interview of Una O’Doherty, Associate Professor of Pathology and Laboratory Medicine at the Hospital of the University of Pennsylvania, Department: Pathology and Laboratory Medicine, Philadelphia, PA, USA

Una kindly agreed to answer a few questions about the recent findings of her laboratory in the area of HIV-1 latency and the development of cellular models of latency.

Alain Lafeuillade: What is the relative role of unintegrated and integrated DNA in HIV persistence?

Una O’Doherty: We think that unintegrated HIV DNA contributes little to persistence since integration is required for efficient production of HIV proteins and infectious virus. Unintegrated HIV DNA is composed of integration competent linear DNA and dead-end circular forms (1 and 2-LTRs). The concept of pre-integration latency exists, but the unintegrated linear form has such a short half-life that it likely does not contribute much to the reservoir. However, since linear unintegrated HIV DNA has a short half-life, detection of this form may reflect recent rounds of replication. While the half-lives of the circular forms are still in question, they do not contribute to persistence as they are dead-end products that cannot integrate into the human genome.

UO: We found that elite suppressors (at least the cohort we measured) have this remarkable pattern of HIV DNA intermediates - they have extremely low levels of integrated HIV DNA with a strikingly large excess of unintegrated HIV DNA (often composed of 2-LTR circular forms). Our hypothesis is that their immune system is playing a role in control of reservoir size. Simply put, integrated HIV DNA may be preferentially cleared because in these patients resting CD4+ T cells with integrated HIV DNA are targeted by HIV specific CD8 T cells. The unintegrated forms on the other hand are likely invisible to the immune system. It may also be that the circular forms accumulate preferentially because they are more stable than the linear unintegrated forms.

AL: Do we have an estimate of the proportion of defective proviruses in the persisting reservoir of patients on long-term cART?

UO: Only a few studies have addressed the amount of defective proviruses in HAART patients. Data from Siliciano’s group suggest that ~99% of proviruses are defective in patients on HAART. We have preliminary data that support Siliciano’s numbers and some evidence that a higher fraction of proviruses may be defective in elite suppressors. We are currently partaking in collaborative studies funded by amfAR to answer this question more definitively.

AL: Several primary cell models of HIV latency have been established (your group, Planelles group, Greene group): how do they compare in terms of reflecting what really happens in vivo?

UO:This is actually one of the fundamental questions facing the latency field and the answer is it’s hard to say. While the field has advanced its knowledge of the latent HIV reservoir, there is still a lot we don’t understand. Thus, at this time, it is hard to determine which model best represents what happens in vivo. For example, there are several different subsets of CD4+T cells that may contribute to HIV reservoirs in patients. Some models (such as the Planelles model and the Siliciano model) exclude some of these CD4+T subsets. On the other hand, the advantage of the Planelles and Siliciano models is the large number of latent cells that are generated making large scale studies much easier. These models are great for screening drugs and for studying more uniform cells biochemically. The benefit of our model is that it maintains the CD4+T cells as they exist in vivo, but does not generate as large a pool of latently infected cells. So there’s a trade off. In the end it really comes down to each model has advantages and only by utilizing several models in a complementary manner can we really begin to see what happens in vivo.

AL: What would be the ‘perfect’ virological tool to assess the changes in HIV reservoirs during eradication trials in patients?

UO: Ideally, we would measure the number of proviruses capable of productive infection using the Infectious Units Per Million (IUPM) assay; however, that method not only requires large numbers of cells in order to make measurements, but the measurement error is also high (because levels are so low) making it often impossible to detect changes in reservoir size. Our data suggests that integrated HIV DNA is often a good surrogate and is sometimes preferable to total HIV DNA. It is much easier to measure than IUPM and we can detect subtle differences in the level of integrated HIV DNA, and therefore the size of the HIV reservoir, after treatment with therapies aimed at eradication. It is estimated that for every infectious integrated form of HIV there are about 100 defective integrated forms. Thus there are 100 times higher levels of integrated HIV DNA than IUPM making it easier to measure integrated HIV DNA more robustly with fewer cells. The question remains whether the proportion of defective viruses is constant over time and within patients. Hopefully, ongoing studies supported by amfAR will help answer that question.